The present invention relates generally to the field of agricultural biotechnology and more particularly to transgenic plants exhibiting enhanced flavor quality and stability, and methods of making the plants.
Methionine (Met) is the precursor for methional, an important flavor compound in various plants such as potatoes, as well as in meats and cheese cracker flavors. The production of methional is thermally induced. It is formed as a result of the interaction of alpha-dicarbonyl compounds, which are formed during the Maillard reaction, with Met through the Strecker degradation reaction. Methional readily decomposes to yield methanethiol, which oxidizes to dimethyl disulfide. Furthermore, derivatives of methionine, such as S-methylmethionine, release dimethyl sulfide that is responsible for the aromas of fish, canned sweet corn, tomato juice and stewing oysters and clams. Methional and S-methyl methionine are heat labile and readily decompose during food processing. Due to high costs of production, these flavor compounds as well as the Met precursor are not added back during food processing.
Met, a sulfur containing amino acid is extremely important in all living organisms. Met serves central roles in metabolism as the initiator tRNA in protein synthesis, as S-adenosylmethionine (SAM), the primary methyl donor for most transmethylation reactions and as a precursor for polyamines and the phytohormone, ethylene in plants. Animals cannot synthesize Met, for this reason it is considered an essential amino acid for animal nutrition.
Due to the nutritional importance of Met, there have been attempts to develop plants with high Met content by genetic engineering. It has been discovered that an abundant seed protein in Brazil Nut (BNP) contains an exceptionally high content of Met (18%) and Cys (8%) (Ampe, et al., Eur. J. Biochem. 159:597-604 (1986); Sun, et al., Eur. J. Biochem 162:477-483 (1987)). Heterologous over-expression of BNP in transgenic tobacco (Altenbach, et al., Plant Mol. Biol. 13:513-522 (1989)) and Brassica (Guercbe, et al., Mol. Gen. Genet. 221:306-314 (1990) ) resulted in a 30% increase in seed Met content. Transgenic plants over-expressing a Met-rich protein such as a sunflower seed albumin (Molvig, et al., Proc. Natl. Acad. Sci USA 94:8393-8398 (1997)) also showed a 94% increase in Met content. An unfortunate property of BNP is that it is a potent allergen in humans with allergies to nuts making the transgenic plants unsuitable for human consumption. See Nordlee, et al., N. Eng. J. Med. 334:688-692 (1996) and Bartolome et al., Allergol. Immunopathol. (Madrid) 25:135-144 (1997). These high-Met proteins are not metabolized to release free Met, however, until consumption. Thus, there is no increase in free Met levels prior to processing and no increase to in methional levels during processing.
Other attempts have focused on deregulation of the Asp family pathway. Transgenic tobacco seeds over-expressing bacterial feedback insensitive aspartate kinase showed a 15.5-fold increase in Thr content but only a 3-fold increase in Met (Shaul, et al., Plant Mol. Biol. 23(4):759-768 (1993); Karchi, et al., Proc. Natl. Acad. Sci. USA 91:2577-2581 (1994). This suggests that the carbon skeleton is not limiting for Met synthesis and that a Met-specific regulation exists. It was also revealed that there was no direct correlation between Lys level and the dihydrodipicolinate synthase (DHPS) activity in transgenic plants over-expressing the feedback-insensitive bacterial DHPS (Ben-Tzvi Tzchori, et al., Plant Mol. Biol. 32:727-734 (1996). The free Lys level was significantly reduced in mature seeds because Lys is efficiently catabolized (Karchi, et al., supra). Further analysis revealed that a Lys degradation pathway was induced in the transgenic plants (Galili, et al., Plant Cell 7:899-906 (1995)). Another attempt to overproduce Met in Arabidopsis was to select for mutants resistant to ethionine, a toxic analog of Met (Alix, Microbiol. Rev. 46(3):281-295 (1982)). Mutants that are resistant to ethionine have been characterized in cultured plant cell lines (Widholm, Can. J. Bot. 54:1523-1529 (1976); Reish, et al., Theor. Appl. Genet. 59:89-94 (1981); Gonzales, et al., Plant Physiol. 74:640-644 (1984); Madison and Thompson, Plant Cell Rep. 7:473-476 (1988)) all of which are reported to accumulate Met. One Arabidopsis mutant termed the mtol showed over-accumulation of soluble Met at the vegetative growth stage but the Met level returned to normal after flowering (Inaba, et al., Plant Physiol. 104:881-887 (1994)).
Hence, a need remains for reliable methods for preparing transgenic plants having increased free met content, and particularly plants that are processed into foodstuffs and food additives.
One aspect of the present invention is directed to transgenic plants of the Solanaceous family e.g., tomato, potato and eggplant, containing at least one non-native nucleic acid that when expressed in the plants, results in increased free methionine levels relative to native free methionine levels. Processing of edible portions of the plant e.g., to make a food product or food additive containing the processed plant portion(s), results in an increase in methional levels compared to methional levels present in a processed edible portion of a wild-type plant. In preferred embodiments, the non-native nucleic acid encodes cystathionine gamma synthase (CGS). In other preferred embodiments, increased free methionine levels in potatoes result from expression of a nucleic acid containing a tuber-specific promoter regulatory unit operably linked (in operative association with) and anti-sense S-adenosyl-methionine synthetase (SAMS)-encoding nucleic acid. In yet other preferred embodiments, a non-native nucleic acid containing a tuber-specific promoter linked to a DNA molecule encoding a SAMS DNA in the sense orientation, that is homologous to the potato. Seed derived from the transgenic plants are also provided.
Another aspect of the present invention is directed to transgenic plants having edible portion(s) that produce methional when processed, such as maize and soybean plant, that contain increased free methionine levels relative to native free methionine levels. Maize is especially preferred. Edible portions of these plants contain increased methional levels compared to methional levels in a processed, wild-type plant. The increased free methionine levels are achieved by expression of a non-native nucleic acid that is other than a nucleic acid encoding a plant CGS, particularly in the seeds of the plants.
Yet another aspect of the present invention is directed to methods of making the aforesaid transgenic plants.
A further aspect of the present invention is directed to making processed products such as foods or food additives containing edible parts thereof that exhibit increased flavor stability and/or quality. The methods entail preparing the aforesaid transgenic plants that contain increased methionine levels, and harvesting and then processing the plant parts, whereupon the processing results in increased methional levels. Products such as foods and food additives that contain the processed plant or parts thereof are also provided.
Yet a further aspect of the present invention is directed to a method for selecting plant cells containing a non-native nucleic acid of interest (e.g., a structural gene encoding a protein of interest), and a selection agent/marker gene combination for use therewith. The method entails transforming plant cells with a chimeric nucleic acid that contains inoperable association, a promoter functional in a plant cell and a first and a second DNA molecule. The first DNA molecule is preferably a structural gene encoding a protein of interest and the second DNA molecule encodes a CGS that permits the selection of a transformed plant cell containing the chimeric nucleic acid molecule by rendering the transformed plant cell resistant to an amount of ethionine that would be toxic to a plant cell that does not express the DNA encoding the CGS. The transformed plant cells are cultured in medium containing ethionine in an amount that would be toxic to plant cells that do not express the CGS-encoding DNA. Plant is cells that grow in the medium are selected. The chimeric nucleic acids, plant cells transformed a therewith, and compositions of matter containing the transformed plant cells in medium containing ethionine are also provided.